The presently disclosed methods relate generally to the field of antibody development or production. In particular, the methods relate to the field of in vitro monoclonal antibody production.
Monoclonal antibodies (mAbs) are highly specific affinity reagents used for detecting and treating diseases. For example, mAbs may be used in localizing biomarkers in tissue, purifying biomarkers from complex substances, and measuring markers leading to diagnosis of diseases cancer in clinical samples (e.g., cancer). The need for mAbs as affinity reagents is continually growing with the advent of multi-analyte detection platforms such as protein microarrays. The advancement of these platforms has yielded the potential for fast, high-throughput analysis of complex samples for small molecules and proteins of interest. The next few years will likely see rapid advancement in the use of these platforms for cancer biomarker discovery and the early-stage diagnosis of different cancers.
Despite the advancement of multi-analyte platforms, the overall performance and usefulness of these approaches depends on the quality of the mAbs used to capture and detect molecules of interest on the microarray surface. Successful development of a microarray requires the screening of many mAbs for affinity, specificity, cross-reactivity, and platform compatibility. For a given target, dozens of mAbs may need to be screened, and thus the techniques used to generate the mAbs must be able to generate a panel of highly diverse antigen-specific (Ag-specific) mAbs. Currently, the means to develop mAbs are limited to a few different strategies, each with limited abilities to generate clonally diverse panels of Ag-specific mAbs.
The standard method for creating monoclonal antibodies to an antigen involves the creation of a fused cell called a “hybridoma.” A hybridoma is produced by fusing together an established tumor cell line, such as a myeloma cell line, and an antibody-producing cell (e.g., a B-lymphocyte) from an animal that has previously been immunized with the antigen. Antibody-producing cells typically are obtained from the animal's spleen, lymph nodes, or lymph tissue (e.g., splenocytes or lymphocytes). These fused cells or “hybrid cells” then are selected and screened to obtain a hybridoma that produces the desired antibody (i.e., a “hybridoma”).
Hybridoma technology has significant disadvantages. Typically, one hybridoma clone may be generated per 105-106 splenocytes fused, thus most of the Ag-specific cells contained within the splenocyte population may be lost. In addition, many of the clones generated may not produce mAbs that recognize the antigen of interest. Furthermore, hybridoma cell lines of interest must be separated through screening and subcloning. The frequency of successful, Ag-specific B cell hybridomas may be on the order of one per 106-108 starting cells. Finally, hybridomas are polyploid and chromosomally unstable. As a result, months of in vitro culture may be required to stabilize each clone and ensure strong mAb production.
An alternative to traditional hybridoma technology is phage display. Phages are viruses that infect bacteria such as E. coli. The phage genome is replicated within the bacteria, translocated to the cytoplasm and packaged into rod-shaped particles, which are then released into the media upon bacterial lysis. The particle coats can be engineered to “display” ligands such as antibodies. Thus large phage libraries containing billions of different antibody genes can be generated, with each phage containing a single antibody gene. These libraries can be screened for binding against any antigen of interest and the desired clone selected. A major advantage of phage display is that it does not require animal immunization. This also may be a primary drawback, because antibodies developed using naïve antibody phage libraries may have affinities that are generally two to three orders of magnitude lower than those of antibodies produced using traditional fusion technology. Increasing the probability of obtaining high-affinity antibodies with phage display requires additional mutagenesis upon clone selection, greatly increasing the naïve library size, or generating libraries from immunized animals. Each option requires extensive development time and expense.
Plasmacytoma technology may be used as an alternative to hybridoma technology and phage display technology. Plasmacytomas are immortalized, antibody producing cells. Plasmacytomas have been obtained by infecting B cells with an immortalizing retrovirus. For example, the ABL-MYC retrovirus has been used to produce plasmacytomas. ABL-MYC is a replication-defective retrovirus, which contains v-abl from the Abelson Murine Leukemia virus (Ab-MuLV) and murine c-myc. ABL-MYC infection stably transforms Ag-specific B cells into plasmacytomas that produce an antibody to a specified target antigen. Antigen-specific plasmacytomas may be obtained by infecting splenocytes from immunized mice with ABL-MYC and then subsequently injecting the infected splenocytes into recipient mice for plasmacytoma and ascites development. This process may provide antibodies against a wide range of antigens. However, clonal diversity may be limited by in vivo clonal selection, plasmacytoma development, and plasmacytoma propagation. For example, one disadvantage of plasmacytoma technology is that clonal diversity may be limited by in vivo ascites development. During plasmacytoma development, many Ag-specific clones may be lost to other clones with more aggressive growth characteristics. In addition, successful Ag-specific plasmacytoma development may depend on strong immune responses from the immunized mice upon antigen challenge. Antigens that elicit weak to moderate immune responses may be unlikely to develop Ag-specific plasmacytomas.
Thus, new methods for obtaining antigen-specific B-lymphocytes are desirable. In particular, new methods for selection and clonal expansion of antigen-specific B-cell populations to produce stable hybridomas and plasmacytomas are desirable.